1. Field of the Invention
The present invention relates to an enzyme that occurs in Rhodococcus species. In particular, the invention relates to an L-amino acid oxidase (L-AAO) from Rhodococcus, and particularly to that of Rhodococcus opacus DSM 42350 as well as to nucleic acids, vectors and microorganisms encoding or expressing L-AAOs. The L-AAOs of the present invention may be used commercially and industrially, for instance, for the production of keto acids or for the purification of D-amino acids from racemic mixtures of amino acids.
2. Description of Related Art
L-amino acid oxidases isolated from snake venom have been intensively studied. These include, for instance, L-amino acid oxidases from Crotalus atrox, see Torii, S., M. Naito, and T. Tsuruo, Apoxin I, a novel apoptosis-inducing factor with L-amino acid oxidase activity purified from Western diamondback rattlesnake venom. J. Biol. Chem. 272 (14):9539-42 (1997); Crotalus adamanteus , see Raibekas, A. A. and V. Massey, Primary structure of the snake venom L-amino acid oxidase shows high homology with the mouse B cell interleukin 4-induced FIG. 1 protein. Biochem. Biophys. Res. Commun. 248(3):476-8 (1998); Calloselasma rhodostoma, see Ponnundurai, G., M. C. Chung, and N. H. Tan, Purification and properties of the L-amino acid oxidase from Malayan pit viper (Calloselasma rhodostoma) venom. Arch. Biochem. Biophys. 313(2):373-8 (1994); Agkistrodon contortrix laticinctus, see Souza, D. H., et al., Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom: preliminary crystallographic data. Arch. Biochem. Biophys. 368(2):285-90 (1999); Bothrops cotiara, see Pessatti, M., et al., Screening of Bothrops snake venoms for L-amino acid oxidase activity, published erratum appears in Appl. Biochem. Biotechnol. 55(3):276 (December 1995); Appl. Biochem. Biotechnol. 51-52:197-210 (1995); Lachesis muta muta, see Sanchez, E. O. and A. Magalhaes, Purification and partial characterization of an L-amino acid oxidase from bushmaster snake (Surucucu Pico de Jaca) Lachesis muta muta venom. Braz. J. Med. Biol. Res., 24(3):249-60 (1991); Pseudechis australis, see Stiles, B. G., F. W. Sexton, and S. A. Weinstein, Antibacterial effects of different snake venoms: purification and characterization of antibacterial proteins from Pseudechis australis (Australian king brown or mulga snake) venom. Toxicon. 29(9):1129-41 (1991).; Ophiophagus hannah, see Ahn, M. Y., B. M. Lee, and Y. S. Kim, Characterization and cytotoxicity of L-amino acid oxidase from the venom of king cobra (Ophiophagus hannah). Int. J. Biochem. Cell. Biol. 29 (6):911-9 (1997); Naja naja kaouthia, see Tan, N. H. and S. Swaminathan, Purification and properties of the L-amino acid oxidase from monocellate cobra (Naja naja kaouthia) venom. Int. J. Biochem. 24(6):976-73 (1992).
L-amino acid oxidases are also found in algae, see Ito, K., K. Hori, and K. Miyazawa, Purification and some properties of L-amino acid oxidase from Amphiroa crassissima Yendo. Hydrobiologica, 151/152:563-569 (1987); Piedras, P., et al., Purification and characterization of an L-amino-acid oxidase from Chlamydomonas reinhardtii. Planta 188:13-18 (1992), the cyanobacteria Synechococcus, see Pistorius, E. K. and H. Voss, Some properties of a basic L-amino-acid oxidase from Anacystis nidulans. Biochim. Biophys. Acta. 611(2):227-40 (1980); fungi, see Kusakabe, H., et al., A new antitumor enzyme, L-lysine alpha-oxidase from Trichoderma viride. Purification and enzymological properties. J. Biol. Chem. 255(3):976-81 (1980); Le, K. H. and V. R. Villanueva, Purification and characterization of epsilon-N-trimethyllysine L-amino oxidase from Neurospora crassa. Biochim. Biophys. Acta. 542(2):288-96 (1978).
They may also be found in certain bacteria, see Cioaca, C. and A. Ivanof, Bacterial amino acid oxidases. I. L-amino acid oxidase and its distribution in bacteria. Arch. Roum. Pathol. Exp. Microbiol. 33(3-4):211-22 (1974); Gamati, S. and J. H. Luong, Production and purification of L-phenylalanine oxidase from Morganella morganii. Bioseparation 2(3):147-54 (1991); Li, Q. S., J. J. Xu, and J. J. Zhong, Production of L-glutamate oxidase and in situ monitoring of oxygen uptake in solid state fermentation of Streptomyces sp. N1. Applied Biochemistry and Biotechnology, 62:243-250 (1997); Koyama, H., Purification and characterization of a novel L-phenylalanine oxidase (Deaminating and decarboxylating) from Pseudomonas sp. P-501. J. Biochem. (Tokyo) 92(4):1235-40 (1982); Brearley, G. M., et al., Purification and partial characterization of a broad-range L-amino acid oxidase from Bacillus corotarum 2Pfa isolated from soil. Appl. Microbiol. Biotechnol. 41(6):670-676 (1994); Bouvrette, P. and J. H. T. Luong, Isolation, purification, and further characterization of an L-phenylalanine oxidase from Morganella morganii. Applied Biochemistry and Biotechnology 48:61-74 (1994). The gene sequences of some L-AAOs are known.
On the other hand, despite the identification of gene sequences for certain types of L-AAOs, it has not been possible to develop an expression system for the quantitative production of L-AAOs. The L-AAO from Crotalus atrox is the only enzyme that it was possible to express at all, by means of active heterologous expression, see Torii, S., et al., Molecular cloning and functional analysis of apoxin I, a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity, Biochemistry 39 (12):3197-205 (2000). The expression was carried out in human embryonal kidney cell cultures and the active enzyme is present in extracellular form. However, the expression of functional L-AAO from Crotalus atrox in E. coli was not successful. A reason for this could be the glycosylation processes or other modifications that generally make the expression of functional, eukaryotic proteins in microbial systems difficult or impossible.
The sequence of the prokaryotic L-AAO from Synechococcus was published for the first time in 1995, see Bockholt, R., et al., Partial amino acid sequence of an L-amino acid oxidase from the cyanobacterium Synechococcus PCC6301, cloning and DNA sequence analysis of the aoxA gene. Biochim. Biophys. Acta. 1264(3):289-93 (1995) and was clearly corrected again in 1998 (gi:3341474). Until now, however, there has surprisingly been no information about the actual N-terminus of the active enzyme.
The presumable L-AAO gene from Bacillus subtilis is probably not complete in the form in which it can be found in databases, since a large portion of the highly conserved FAD binding site is missing and therefore, the presumed L-AAO gene of Bacillus subtilis as shown in databases is probably not complete, see Vallon, O., New sequence motifs in flavoproteins: Evidence for common ancestry and tools to predict structure, Proteins 38(1):95-114 (2000). Similarly, a gene product from Chromobacterium violaceum has not yet been proven to be an intact, expressible L-AAO sequence (gi:5802874). In the absence of such information, the search for, and discovery of, a suitable expression system for these prokaryotic L-AAOs has been complicated and unproductive.
Previously known L-AAOs have been used in biosensor technology, see Liu, J. and J. Wang, Remarkable thermostability of bioelectrodes based on enzymes immobilized within hydrophobic semi-solid matrices [In Process Citation]. Biotechnol Appl Biochem. 30(Pt 2):177-83 (1999); Varadi, M., et al., Determination of the ratio of D- and L-amino acids in brewing by an immobilized amino acid oxidase enzyme reactor coupled to amperometric detection. Biosens Bioelectron. 14(3):33540 (1999); Sarkar, P., et al., Screen-printed amperometric biosensors for the rapid measurement of L- and D-amino acids. The Analyst 124:865-870 (1999); Lee, Y. C. and M. H. Huh, Development of a biosensor with immobilized L-amino acid oxidase for determination of L-amino acids. Journal of Food Biochemistry 1999:173-185 (1998).
On a small preparative scale, various L-AAOs have been investigated for the transformation of L-lysine derivatives, see Hanson, R. L., et al., Transformation of N epsilon-CBZL-lysine to CBZ-L-oxylysine using L-amino acid oxidase from Providencia alcalifaciens and L-2-hydroxyisocaproate dehydrogenase from Lactobacillus confusus. Appl. Microbiol. Biotechnol. 37(5):599-603 (1992), for the transformation of L-methionine, Takahashi, E., et al, D-methionine preparation from racemic methionines by Proteus vulgaris IAM 12003 with asymmetric degrading activity. Appl. Microbiol. Biotechnol. 47(2):173-179 (1997), and for the transformation of xcex2-N-methylamino-L-alanine, Hashmi, M. and M. W. Anders, Enzymatic reaction of beta-N-methylaminoalanine with L-amino acid oxidase. Biochim. Biophys. Acta. 1074(1):36-94 (1991).
Accordingly, in view of the problems with existing L-AAOs, one object of the present invention is to provide an L-AAO that is easy to produce and that can be produced in a quantity suitable for commercial and industrial uses, such as for the production of keto acids or for the purification of D-amino acids.
The present invention provides an L-AAO from Rhodococcus, preferably from Rhodococcus opacus, and most preferably from Rhodococcus opacus, strain DSM43250. Rhodococcus species within the scope of the present invention, are bacteria that can be classified in Group 22, Subgroup 1 according to Bergey""s Manual of Determinative Bacteriology, 9th Ed., Ed.: Hensyl, W. R., Williams and Williams, Baltimore (1994).
L-amino acid oxidases (L-AAO) from Rhodococcus species are provided. Most advantageously the present invention provides an L-AAO from Rhodococcus opacus DSM43250 comprising SEQ ID NO: 2 and biologically active fragments (e.g. fragments with an L-AAO activity) of this sequence.
Yet another object is to provide gene sequences, vectors, microorganisms or host cells that encode or express Rhodococcus L-AAOs or biologically active fragments of such L-AAOs. Moreover, it is also an object of the invention to provide such gene sequences and their complements as probes and primers for the identification or production of other L-AAO sequences.
Other aspects of the invention include methods of using Rhodococcus L-AAOs to produce keto acids or for the isolation or purification of D-amino acids by the removal of L-amino acids. The enzymes according to the invention are particularly well suited for technical use and production of enantiomer-concentrated amino acids.